A straightforward and versatile approach to permanently introduce new physical and biological properties into DNA by irreversible chemical modification of plasmid would be a useful tool in the design and implementation of synthetic gene delivery systems for gene therapy applications. However, all of the currently available methods for chemically modifying plasmid damage the DNA and interfere with its transcriptional activity. The purpose of this grant is to provide proof of concept for a non- damaging approach to irreversibly modify DNA. The method takes advantage of the property of some peptide nucleic acids (PNA) to hybridize to duplex DNA in a very high affinity and sequence-specific manner. To demonstrate this gene chemistry approach a fluorescent PNA conjugate will be hybridized to a specific site cloned into a plasmid. The fluorescent signal from the PNA will be utilized to determine the stoichiometry, specificity, binding affinity and reversibility of the PNA bound to its target sequence. Transcriptional activity of an expression plasmid with and without PNA will be compared. The use of the fluorescent PNA in association with a plasmid encoding green fluorescent protein will be evaluated as a dual fluorescent system for simultaneous quantitative monitoring of plasmid DNA biodistribution and plasmid based gene expression. PROPOSED COMMERCIAL APPLICATION: The phase II grant proceeds will be used to design the next generation of pGeneGrip(TM) Vectors which will contain Nuclear Localization Peptides, Endosomal Lytic Peptides, Receptor Specific Ligands and Transcription Activators. The resources from the phase II grant application will be used to make the pGeneGrip(TM) Vectors available to research scientists performing DNA bioavailability studies in vitro and in vivo.